Tuberculosis has been declared a WHO global health emergency, and the recent emergence of multidrug (MDR) and extensively drug resistant (XDR) TB has underscored the urgent need for new TB drugs. We hypothesize that HTS will identify probes which chemically sensitize Mycobacterium tuberculosis (M. tb.) to the activity of existing 2-lactam and macrolide antibiotics. The identification of such probes could lead to combination antibiotics, similar to amoxicillin-clavulanate, which have potency against M. tb. This proposal seeks access to the HTS resources provided by the Molecular Libraries Screening Center Network (MLSCN) with the goal of identifying sensitizing probes, identifying their molecular targets, and finding candidate agents as potential lead structures for further study. An HTS-ready, fluorescence-based whole cell screening assay is proposed that will utilize virulent M. tb. in combination with optimized concentrations of imipenem (IMI, representing the 2-lactam class) and clarithromycin (CLA, representing the macrolide class). The following aims are proposed to conduct an efficient HTS evaluation for probes that are synthetically lethal against M. tb. in the presence of 2-lactam and macrolide antibiotics: to transfer the M. tb./adjuvant drug susceptibility protocol to the designated screening center to reproduce, miniaturize, and automate a corresponding HTS assay that will be used to screen the MLSCN compound library for sensitizing probes, to provide technical support, and to conduct secondary assays confirming the potency and specificity of identified probes. PUBLIC HEALTH RELEVANCE: Tuberculosis has been declared a WHO global health emergency, and the recent emergence of multidrug (MDR) and extensively drug resistant (XDR) TB has underscored the urgent need for new TB drugs;indeed, many antibiotics such as those from the penicillin-, erythromycin-, and tetracycline-families are not clinically useful in the treatment of TB either because of poor bacterial penetration or drug degradation by the microbe. Our preliminary studies reveal that specific mutations can sensitize Mycobacterium tuberculosis (M. tb.) to penicillin and erythromycin, thus supporting the hypothesis that new drugs may be identified that would also sensitize the microbes to the activity of these familiar classes of antibiotics. We have developed a microtiter plate assay to seek compounds which amplify the activity of key members of the penicillin- and the erythromycin-class against M. tb., and we propose to transfer this assay to an appropriate MLSCN laboratory for high-throughput screening to identify potential lead compounds.